Cloning, Expression, and Characterization of Recombinant Fab

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J. Agric. Food Chem. 1998, 46, 3381−3388

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Cloning, Expression, and Characterization of Recombinant Fab Antibodies against Dioxin Nanju Lee,* Carol K. Holtzapple, and Larry H. Stanker Food Animal Protection Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 2881 F&B Road, College Station, Texas 77845-9594

Using two hybridoma cell lines (DD1 and DD3) secreting anti-dioxin monoclonal antibodies as a source for messenger RNA and cDNA, light and heavy chain gene fragments of Fab domains were amplified by the polymerase chain reaction (PCR). The amplified gene fragments were cloned into the pFabUSDAI vector for expression of recombinant Fab antibodies in Escherichia coli. Expression of the soluble and functional recombinant Fab antibodies (designated rFab1-1 and rFab3-3) was confirmed by an indirect immunoassay using dioxin conjugated to rabbit serum albumin. On the basis of these rFabs, two competitive inhibition immunoassays using 2,3,7,8-tetrachlorodibenzo-pdioxin (2,3,7,8-TCDD) as a competitor were developed. The concentration of 2,3,7,8-TCDD required to inhibit color development by 50% (IC50) determined from the dose response curves for rFAB1-1 and rFAB3-3 were 10.4 ( 2.4 and 12.2 ( 6.0 ng/mL, respectively. The binding properties of both rFab antibodies for other chemically related compounds were relatively similar to those of their respective monoclonal antibodies and enzymatically derived Fab fragments. Keywords: Dioxin; recombinant antibody; cloning antibody genes; Fab; expression vector; Escherichia coli; immunoassay INTRODUCTION

The development of hybridoma technology in 1975 (Kohler and Milstein, 1975) allows the production of an unlimited supply of homogeneous antibody. Since then, monoclonal antibodies have been used successfully and extensively in many areas of science. In environmental diagnostics, sensitive immunoassays using monoclonal antibodies have been developed for numerous compounds. These include dioxin (Stanker et al., 1987), atrazine (Giersch and Hock, 1990), metolachlor (Schlaeppi et al., 1991), paraquat (Niewola et al., 1986), and picloram (Deschamps et al., 1990). Monoclonal antibody production requires fusion of differentiated B-lymphocytes and myeloma cells, and an extensive and laborious screening process must be performed to obtain the desired hybridomas. The drawback of the technology, however, is that the selection is limited to the more prevalent populations of clones that may exhibit lower affinity for the analyte. Clones with high affinity may not have the opportunity to be selected due to either the screening strategy itself or perhaps chromosomal instability of specific clones. More importantly, the properties of the monoclonal antibody cannot be readily altered, and improvements can be achieved only through conventional approaches, for example, by synthesis of a new immunogen. Rapid developments in molecular biology and protein engineering now make it possible to engineer “tailormade” polypeptides or proteins such as immunoglobulins with either specific or improved functionality. Since the early reports describing recombinant antibody technology in the mid 1980s, significant progress has been * Author to whom correspondence should be addressed [telephone (409) 260-9306; fax (409) 260-9332; e-mail lee@ usda.tamu.edu].

made, especially in the design of expression vectors, phage display systems, isolation of heavy and light chain encoding genes by polymerase chain reaction (PCR), and various expression systems for bulk production. Different antibody fragments such as Fv (Skerra and Plu¨ckthun, 1988), single-chain Fv (scFv; Bird et al., 1988), Fab (Better et al., 1988), (Fab′)2 (Carter et al., 1992), miniantibodies (Sollazzo et al., 1995), and diabodies (Holliger et al., 1993) have now been expressed. Also, antibody molecules fused to reporter enzymes or affinity tags can be easily engineered. Furthermore, modification of an existing antibody or design of a new one can be achieved at the molecular level via sitedirected mutagenesis, chain shuffling, or complementarity-determining region (CDR) grafting. Antibody engineering technology was developed initially for medical and biochemical diagnostics and for therapeutic applications. More recently, several research groups have examined the potential of this technology for developing more useful antibodies for environmental diagnostics. Although many recombinant antibodies have been engineered for biomedical and clinical applications, only a few recombinant antibodies have been reported for environmental diagnostics. These include recombinant Fab antibodies for atrazine (Ward et al., 1993) and diuron (Karu et al., 1994; Scholtholf et al., 1997) and scFv fragments for s-triazine (Kramer and Hock, 1996), parathion (Garrett et al., 1997), and cyclohexanedione (Webb et al., 1997). Although the technology has proven to be useful for generating more appropriate antibody molecules, many researchers have found technical difficulties in synthesizing recombinant antibodies routinely. The inability to generate high-affinity recombinant antibodies from synthetic combinatorial libraries caused a number of researchers to use hybridomas secreting relevant mono-

S0021-8561(98)00285-4 This article not subject to U.S. Copyright. Published 1998 by the American Chemical Society Published on Web 07/18/1998

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clonal antibodies as the starting point for developing recombinant antibodies. Even with the use of hybridomas, problems arose due to unequal production of the light and heavy chains during PCR amplification, contamination of high-affinity clones capable of recognizing hapten in solution with nonspecific clones during the panning step, and poor recombinant protein production and recovery during expression. A number of alterations in the methodology have improved the technology. These include development of more effective primer mixes for amplification of both the heavy and light chains (Zhou et al., 1994), use of an immunomagnetic separation method to reduce contamination of high-affinity clones (Kramer and Hock, 1996), and development of better purification protocols (Byrne et al., 1996; Kipriyanov et al., 1997) for improved recombinant antibody production. Despite the difficulties, antibody engineering has the potential to produce more effective antibodies for environmental analyses. Dioxins are environmental contaminants that are considered carcinogenic and that can pose a potential threat to human health if adulterated food is consumed. Milk was found to be contaminated with polychlorinated dibenzo-p-dioxin and polychlorinated dibenzofurans (Ryan et al., 1991; Schuler et al., 1997). More recently, chicken carcasses were contaminated with dioxin; the source of contamination was the clay used in the feed (Food Chemical News, 1997). These incidents demonstrate that dioxins can find their ways into the food chain through contaminated raw materials. Conventional instrumental analyses require laborious multistep cleanup procedures and are very expensive. In an effort to provide simplified and routine analyses, a number of immunoassays for dioxin have been developed previously (Albro et al., 1979; Kennel et al., 1986; Stanker et al., 1987). A panel of monoclonal antibodies developed by Stanker et al. (1987) provides sufficient sensitivity, specificity, and accuracy for analysis of dioxin in industrial samples and in soil (Vanderlann et al., 1988; Watkins et al., 1989). Two of the monoclonal antibodies, DD1 and DD3, have been fully characterized (Stanker et al., 1995). We have cloned the heavy and light chain genes, obtained from the DD1 and DD3 hybridoma cell lines that secrete anti-dioxin monoclonal antibodies, into the pFabUSDAI vector. Two recombinant Fab fragments (rFab) were expressed in Escherichia coli. Both of the rFabs displayed competitive inhibition with 2,3,7,8tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) in an indirect ELISA. We chose to express Fab fragments rather than scFv for several reasons. A number of examples, including previous experience in our laboratory, indicated that scFv fragments are likely to be less stable than Fab fragments, partly due to the lack of intramolecular disulfide bonds (Reiter et al., 1996). The constant domains contribute greatly to the interaction between the light and heavy chain, thus increasing the architectural stability of the antibody molecule. For environmental diagnostics, recombinant antibodies are required that are sufficiently stable in various matrixes for analysis. Thus, Fab fragments would be more useful as analytical reagents. Here we report the construction of an expression vector, cloning and expression of recombinant Fab fragments, characterization of these fragments for sensitivity and specificity, and comparison of their binding properties with those of parent antibodies and enzymatically derived Fab fragments.

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Figure 1. Map of pFabUSDAI vector containing a dicistronic operon for expression of anti-dioxin rFab. This construct consists of a T7lac promoter (PT7 lac), pelB regions for secretion of light and heavy chain fragments in the periplasmic space, ribosomal binding sites (rbs), a polyhistidine tag (6x his) for detection and purification, and various restriction enzyme sites for insertion of light and heavy chain genes (cloning sites are indicated by the dashed lines). MATERIALS AND METHODS Reagents. Dibenzodioxin (DD), 2,3,7,8-tetrachloro-DD (TCDD), and 3,3′,4,4′-tetrachlorobiphenyl (TCBP) were obtained from Chem Service Chemicals (West Chester, PA). 2,4,5-Trichlorophenol and 2,5-dichloronitrobenzene were purchased from Aldrich Chemical Co. (Milwaukee, WI). Both γand δ-hexachlorocyclohexane were obtained from Sigma Chemical Co. (St. Louis, MO). Construction of a Recombinant Fab Antibody Expression Vector. The pFabUSDAI expression vector containing appropriate restriction endonuclease sites for cloning purposes was constructed from the existing vectors pET22b+ (Novagen) and pHEN (a generous gift of H. R. Hoogenboom) as follows. The EcoRI, NcoI, and NotI sites in pET22b+ were removed by sequential Klenow fill-in and religation reactions. An EcoRI site was then introduced between the HindIII and XhoI sites in the vector using an adapter. A 6x histidine site followed by a stop sequence (UGA) was added after the SacI site so that the recombinant product could be easily purified by metal chelate affinity chromatography. Finally, the HindIII-EcoRI sequence of pHEN containing a ribosomal binding site, pelB leader sequence, and the gene encoding the phage gIII protein was inserted into the HindIII-EcoRI site of the altered pET22b+. A diagram of the pFabUSDAI expression vector showing the sites of insertion of the antibody genes is shown in Figure 1. Cloning of Recombinant Fab Fragments into the Expression Vector. Messenger RNA was isolated from DD1 and DD3 hybridoma cell lines (Micro-Fast Track kit; Invitrogen, CA), which secreted monoclonal antibodies against the dioxins (Stanker et al., 1987). First-strand cDNA was synthesized from the mRNA using an oligo-d(T) primer (FirstStrand cDNA Synthesis kit; Pharmacia Biotech, Uppsala, Sweden), and the immunoglobulin heavy and light chain genes were amplified from cDNA by PCR using Taq DNA polymerase and specific oligonucleotide primers with the desired restriction sites. To produce Fab fragments, the primers were designed to anneal to the 5′-sequences of framework region 1 of the variable regions and the 3′-sequences of the constant regions. Heavy chain genes were reamplified using a set of second primers designed to attach an additional endonuclease cleavage site (SfiI). The sequences of 5′-primers are as follows (the underlined sequences indicate restriction sites): for DD1 heavy chain, 5′-AGC CGG CCA TGG CCC AGG TCC AAC TGC AGC AGC-3′ (designated 5′DD1H-NcoI) and 5′-ACT CGC GGC CCA GCC GGC CAT GGC CCA GGT CCA A-3′ (5′DD1HSfiI); for DD1 light chain, 5′-ATG GCC ATG CAT GGT GAT GTT GTG ATG ACC CAA GC-3′ (5′DD1-NsiI); for DD3 heavy chain, 5′-GGC CAT GGC CGA CGT AAA CCT GGT GGA GTC3′ (5′DD3H-NcoI) and 5′-ACT CGC GGC CCA GCC GGC CAT GGC CGA CGT AAA-3′ (5′-DD3H-SfiI); for DD3 light chain gene, 5′-ATG GCC ATG CAT GGT GAC ATC CTG ATG ACC CAA TC-3′ (5′DD3L-NsiI). The sequences for 3′-primers are 5′-GTC GAT GCG GCC GCA ATT TTC TTG TCC ACC TTG3′ (3′CH1-NotI) for the heavy chains and 5′-TCG ACG GAG CTC ACA CTC ATT CCT GTT GAA-3′ (3′LC-SacI) for the light chains. PCR was performed for 30 cycles as follows: denaturation at 94 °C for 1 min, annealing at 55 °C for 2 min, and extension at 72 °C for 2 min. PCR products exhibiting a size of approximately 700 bp were excised from agarose gels and purified using microspin columns (Pharmacia, Piscataway, NJ).

Recombinant Fab Antibodies against Dioxin Both the PCR products and the pFabUSDAI vector were digested with the appropriate pairs of restriction enzymes. The digested heavy and light chain DNA fragments were directionally cloned into the vector, and the ligated products were used to transform E. coli NovaBlue cells (Novagen, Madison, WI). Plasmid DNA was isolated from individual colonies and digested with appropriate restriction enzymes, and positive clones were identified by the presence of an approximately 700 bp insert. Expression of Recombinant Fab Fragments to Dioxin. For expression of recombinant Fab fragments, E. coli BL21(DE3) and BL21(DE3)pLysS strains (Novagen) were transformed with plasmid DNA from the positive clones. Transformed cells were plated onto LB agar plates containing carbenicillin (50 µg/mL) for the BL21(DE3) strain or carbenicillin (50 µg/mL) and chloramphenicol (20 µg/mL) for the BL21(DE3)pLysS strain. Plates were incubated at 37 °C overnight, and then each colony was transferred to 5 mL of LB medium containing appropriate antibiotics. Cultures were incubated overnight at 37 °C with continuous shaking. Five hundred microliters of culture was then transferred to 50 mL of LB medium containing appropriate antibiotics and the incubation continued at 30 °C until OD600 ) 0.8-1.0. Cells were induced by adding IPTG to final concentrations of 0.4 and 1 mM, for BL21(DE3) and BL21(DE3)pLysS strains, respectively, and incubating for 3 h or overnight at 30 °C. Preparation of Periplasmic Extract. Periplasmic extract was prepared using a method described by Polymenis and Stollar (1995). Cells were harvested by centrifugation at 5000g for 10 min. Supernatant was collected in a separate tube, and the bacterial pellet was resuspended in cold lysis buffer (200 mM Tris-HCl, 0.5 mM EDTA, 0.5 M sucrose; 10 µL/10 mL cell culture). The resuspended bacterial pellet was added to a tube containing 12 mL of cold purified water and 4 mL of cold lysis buffer. The sample was mixed well and incubated on ice for 30 min. Periplasmic extract was obtained by centrifuging the suspension at 15000g for 10 min at 4 °C and collecting the supernatant. Culture supernatant and periplasmic extract were filtered through a 0.22 µm cellulose acetate membrane and stored at 4 °C until further purification. Purification of Recombinant Fab Antibodies. Recombinant Fab fragments were purified either by cobalt immobilized metal affinity chromatography (IMAC, Clontech, CA) or protein G-Sepharose affinity chromatography. For IMAC purification, 20 mL of culture medium or periplasmic extract was added to pre-equilibrated resin in a gravity flow column and gently agitated for 20 min. Periplasmic extracts were initially dialyzed against Tris-buffered saline (TBS; 20 mM Tris-HCl and 100 mM NaCl, pH 8.0) to remove EDTA. The suspension was centrifuged at 700g for 5 min, and the supernatant was discarded. The resin was washed three times with 10 mL of IMAC wash buffer (20 mM Tris-HCl, 100 mM NaCl, and 10 mM imidazole, pH 8.0). Polyhistidine-tagged antibody was eluted after agitating with 2 mL of IMAC elution buffer (20 mM Tris-HCl, 100 mM NaCl, and 50-100 mM imidazole, pH 8.0) for 10 min. Preparation of Fab Fragments from the Parent Antibodies. Fab fragments were produced by papain digestion of purified IgG (Andrew and Titus, 1991). The papain (2.5 mL of 0.1 mg/mL solution in PBS containing 0.02 M EDTA and 0.02 M cysteine) was added to an equal volume of antibody solution (1.6 mg/mL for DD1 and 2.2 mg/mL for DD3). The mixtures were incubated at 37 °C for 24 h, and enzyme digestion was stopped by adding iodoacetamide to a final concentration of 0.03 M. The solution was dialyzed against PBS for 3 days and purified on a protein G-Sepharose affinity column. The papain digestion was assessed using a polyacrylamide gel electrophoresis (SDS-PAGE) by the disappearance of a band at ∼50 kDa that corresponded to the reduced intact heavy chain. DNA Sequencing. The cloned antibody genes were sequenced using a commercial kit (Sequenase 2.0, Amersham, Arlington Heights, IL) and reverse primers designed to bind either to the framework 2 regions or to the 5′-end of the constant regions. The sequences of the primers are as fol-

J. Agric. Food Chem., Vol. 46, No. 8, 1998 3383 lows: for DD1 heavy chain framework 2, 5′-CCA ATC CAC TCA AGG CCT TG-3′; for DD1 light chain framework 2, 5′ATA GAT TAG GCG TTT TGG AG-3′; for DD3 heavy chain framework 2, 5′-TGC GAC CCA CTC CAG CCT CT-3′; for DD3 light chain framework 2, 5′-ATA GAT CAG GCC CTT AAA TG-3′; for the constant region of the heavy chain, 5′-CAG GGG CCA GTG GAT AGA C-3′; and for the constant region of the light chain, 5′-CTG CTC ACT GGA TGG TGG GA-3′. Electrophoresis. Gel Electrophoresis. SDS-PAGE was performed using either 4-20% gradient gels or 12% acrylamide gels in a Bio-Rad Mini Protein II gel electrophoresis apparatus at 200 V for 40-60 min (Bio-Rad, Hercules, CA). The protein bands were stained with either Coomassie Bright Blue R or silver nitrate for visualization. Immunoblotting. Total proteins from SDS-PAGE were transferred to a poly(vinylidene difluoride) (PVDF) membrane using a semidry immunoblotting apparatus (Bio-Rad). The membrane was blocked with 1% BSA-PBS/T (1% bovine serum albumin in PBS containing 0.01% Tween 20) for 1 h. Following three washes with PBS/T, the membrane was incubated with goat anti-mouse antibody conjugated to horseradish peroxidase (1/1000 in 1% BSA-PBS/T) for 1 h. Unbound conjugate was removed by incubating the membrane in PBS/T three times for 10 min each time. 3,3′,5,5′-Tetramethylbenzidine (1-Step TMB blotting substrate, Pierce, Rockford, IL) was added for color development. All incubations were performed with constant agitation. For detection of the polyhistidine tag, the membrane was incubated for 1 h in mouse anti-polyhistidine monoclonal antibody (diluted 1/3000 in 1% BSA-PBS/T, Sigma) prior to incubation with HRPlabeled goat anti-mouse Fc. Indirect Enzyme Immunoassays. Detection of 6x HisTagged Recombinant Fab. Wells were coated with 0.5 µg/well of 1-N-(adipamino)-3,7,8-trichlorodibenzo-p-dioxin conjugated to rabbit serum albumin (tri-CDD-A-RSA; Stanker et al., 1987). After unbound sites were blocked with blocking buffer (3% nonfat dried milk in PBS, pH 9), 100 µL of supernatant or periplasmic extract was added to each well and incubated for 2 h at room temperature. Wells were washed 10 times with washing buffer (0.1% Tween 20) and 100 µL of mouse antipolyhistidine monoclonal antibody (diluted 1/3000 in 1% BSAPBS/T) was added to each well. After 1 h of incubation at room temperature, unbound antibodies were removed by washing as previously described, and 100 µL of goat anti-mouse conjugated to horseradish peroxidase (diluted 1/1500 in 1% BSA-PBS/T) was added and incubated for 1 h. Washing was performed to remove unbound conjugate, and then 150 µL of K-blue substrate (ELISA Technologies, Lexington, KY) was added and incubated for 30 min. Color development was stopped by adding 50 µL of 2 M sulfuric acid. Absorbance was read at both wavelengths, 450 and 655 nm. Competitive Indirect Enzyme Immunoassay To Detect 2,3,7,8TCDD and Related Compounds. An indirect immunoassay using a solid-phase antigen format was used for the competitive inhibition studies. Wells were coated with coating antigen at 0.25 µg/well. After incubation with blocking buffer, 100 µL of dioxin standard and 100 µL of purified antibody solution were added to each well and incubated for 1.5 h. Wells were washed three times with washing buffer using a microwell plate washer (Bio-Rad). Dioxin waste generated from this step was retained in a waste bottle for appropriate disposal. Goat anti-mouse conjugated to HRP (100 µL per well) was incubated for 1 h, and the unbound conjugate was removed by washing. Color development was performed as described earlier, and absorbance was read at both 450 and 655 nm. Preparation of Dioxin Standards. Dioxin standards were prepared as described by Vanderlaan et al. (1988) with some modifications. 2,3,7,8-TCDD in toluene (10 µg/mL) was dried under nitrogen and redissolved in an equal volume of methanol containing 1% Tween 20. The solution was diluted 1/10 with 1% BSA-PBS/T to obtain 1 µg/mL and then serially diluted 1/3 in the same buffer to give 333, 111, 37, 12, 4, 1.37, 0.46, and 0.15 ng/mL. The final concentrations of methanol and Tween 20 in microwells did not exceed 10 and 0.1%, respectively. Other chlorinated chemicals were prepared in a similar

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Figure 2. Agarose gel electrophoresis of heavy and light chain genes: (A) Lane 1 is DD1 light chain gene amplified from cDNA using 5′DD1L-NsiI and 3′LC-SacI primers; lane 2 is DD1 heavy chain gene amplified using 5′DD1H-NcoI and 3′CH1-NotI primers; lane 3 is DD3 light chain gene amplified using 5′DD3L-NsiI and 3′LC-SacI primers; and lane 4 is DD3 heavy chain gene amplified using 5′DD3H-NcoI and 3′CH1NotI primers. (B) Lane 1 is DD1 heavy chain reamplified using 5′DD1H-SfiI and 3′CH1-NotI primers; and lane 2 is DD3 heavy chain re-amplified using 5′DD3H-SfiI and 3′CH1-NotI primers. DNA molecular weight markers in bp are indicated to the left of each panel. manner. Suitable protective clothing, gloves, and eye protection were worn when dioxin was handled, and the immunoassays were performed in an approved fume hood. The generation of waste material containing dioxin and its congeners was kept to a minimum. The disposal of the solid and liquid waste that was generated was handled by a hazardous waste broker adhering to all federal, state, and local regulations. RESULTS AND DISCUSSION

Cloning and Expression of Recombinant Fab Fragments to Dioxin. The use of hybridoma cell lines that secrete dioxin monoclonal antibodies as a source for mRNA provided several advantages for our studies. First, the DNA sequences from the cDNA of DD1 and DD3 have been published (Recinos et al., 1994; Accession Nos. in EMBL database: DD1-γ Z21788, DD3-κ Z19575, DD3-γ X58884, and DD3-κ X59052), which enabled specific primers to be designed. Second, immunoassays using these monoclonal antibodies were fully developed and characterized. This provided a helpful reference regarding the affinity of these antibodies, thus permitting a quick assessment of and comparison with the parent antibodies to determine whether correct folding of the light and heavy chains had been achieved. Finally, the published molecular modeling data describing the antigen binding sites of these antibodies (Stanker et al., 1995) will be beneficial when mutagenesis is considered for future study. To prepare heavy and light chain genes, mRNA isolated from DD1 and DD3 hybridoma cell lines was used to synthesize first-strand cDNA using a random primer. The heavy and light chain genes were amplified by PCR using primers complimentary to the 5′-end of framework 1 and the 3′-end of constant regions. The agarose gel electrophoretic analyses of the heavy and light chain gene PCR products are shown in Figure 2. Each primer contained a specific restriction enzyme site for directional cloning. For the light chain gene cloning, the primers were designed to contain sequences complimentary to SacI and NsiI. Our initial attempts to use NcoI for directional cloning of heavy chain genes were unsuccessful. We found two NcoI sites present in the variable region of the DD3 heavy chain and also in the constant regions of both heavy chains. The fre-

Lee et al.

quency of occurrence of NcoI sites in mouse heavy and light chain variable regions is reported to be 6.2 and 5.6%, respectively, and occurrence of SfiI sites has not been reported for mouse heavy and light chain variable regions (Ge et al., 1995). Thus, both DD1 and DD3 heavy chain genes were initially amplified with primers containing NcoI and NotI and then reamplified using primers containing SfiI and NotI. Light chain genes were cloned into pFabUSDAI, and the vectors were used to transform E. coli Nova Blue cells. Positive clones were determined by isolating plasmid DNA followed by restriction enzyme digestion and electrophoresis of the digested products. One clone containing the DD1 light chain gene and one containing the DD3 light chain gene were selected for cloning the heavy chain genes. The final clone carrying the DD1 light and heavy chains is designated pRFAB1-1, and the clone carrying the DD3 light and heavy chains is designated pRFAB3-3. The PCR-amplified antibody gene sequences in these clones were identical to the published DNA sequences, indicating that no errors were introduced during PCR amplification. Recombinant Fab antibodies were initially expressed by inducing the transformed BL21(DE3)pLysS strain with 1 mM IPTG. The induction was performed in 50 mL batches for 3 h or overnight at 30 °C with continuous shaking. The lower temperature during induction is an essential factor for the expression of soluble recombinant antibodies, because it allows expression and transport of expressed proteins into the periplasmic space to occur at a slower rate (Schein and Noteborn, 1988; Takagi et al., 1988). Overexpression of recombinant antibodies can result in the formation of insoluble aggregates. Also, it has been noted that an antibody is more likely to fold correctly at a lower temperature. Cosecretion of both light and heavy chains into the periplasmic space was facilitated by the use of the pelB signal sequence placed immediately upstream of each cloning site in the expression vector. Secretion of the rFabs into the bacterial periplasm is preferred, because the periplasm contains disulfide isomerase. This enzyme enhances the formation and rearrangement of disulfide bonds (Bardwell et al., 1991) and assists in the correct folding of proteins. Antibody domains expressed in a nonoxidative environment such as the cytoplasm require in vitro refolding to form functional recombinant antibodies. Secretion of both light and heavy chains into the periplasmic space has an added advantage of eliminating in vitro refolding of the expressed recombinant domains. Detection of rFab1-1 and rFAB3-3 was performed using both indirect ELISA and immunoblotting experiments. The ELISA results indicated that both the culture supernatant and periplasmic extract contained recombinant Fab fragments. rFab1-1 samples showed strong antigen binding as determined from the color development, whereas rFab3-3 samples showed weak binding. To obtain sufficient quantities for the competitive inhibition study, periplasmic extract was initially purified by Talon metal affinity chromatography. A larger quantity of both culture supernatant and periplasmic extract was purified by a protein G-Sepharose affinity chromatography. Protein concentrations of 5.9 and 0.04 µg/mL of induced cell culture were recovered from the protein G purification for rFab1-1 and rFab33, respectively. Since unpaired heavy chain could be present in these solutions, these concentration values

Recombinant Fab Antibodies against Dioxin

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Figure 3. (A) Immunoblot of reducing gel electrophoresis of recombinant Fab fragments in a time course study. Lane 1 is the rFab1-1 expression at 0 h, lane 2 is the rFab1-1 expression at 1 h, lane 3 is the rFab1-1 expression at 2 h, and lane 4 is the rFab1-1 expression at 3 h. Similarly, the rFab3-3 expression at 0, 1, 2, and 3 h is shown in lanes 5, 6, 7, and 8, respectively. The bands shown are approximately at 25 kDa. (B) Indirect ELISA data showing an increase in color development (A280) with induction time (in h).

should be regarded as relative concentrations for the rFab1-1 and rFab3-3 expressions. The amount of the rFab3-3 antibody (4 µg/well) required to produce a similar color development in an ELISA was 100 times higher than that of rFab1-1 (0.04 µg/well). The ELISA titration data indicated that the amount of recombinant Fab antibodies produced by BL21(DE3) strain was similar to that produced by the pLysS strain. No antigen binding was observed for cell supernatant samples, which indicates BL21(DE3) was less prone to cell lysis during induction. To determine the optimum time to harvest, a time course study was undertaken and the results are summarized in Figure 3. An increase in the rFab concentration with time was observed in both indirect ELISA and the immunoblot. From the band intensity of the immunoblot, it was obvious that the light chain was expressed in a larger quantity than the heavy chain. This observation was more prominent in the first 2 h, and expression appeared to reach optimum after 3 h of induction (i.e., the intensities of the ∼25 kDa bands were similar for 3 h and overnight incubations). Stoichiometric expression of heavy and light chains is important for the formation of functional recombinant Fab antibody. The pFabUSDAI vector has been constructed to arrange heavy and light chain genes as a dicistronic operon controlled under one T7lac promoter. One explanation for the greater quantity of the light chain than that of the heavy chain would be that transcription by the T7lac promoter may be more efficient for the light chain gene, because it was cloned proximal to the T7lac promoter. Comparison of rFabs to Parent Antibodies and Fab Fragments Derived from the Parent Molecules for Assay Sensitivity. Initial detection of rFab was performed using an indirect immunoassay with triCDD-A-RSA as a coating antigen. To detect the expression of rFab fused to a polyhistidine tag, an antipolyhistidine antibody was incubated prior to the labeled antibody for color development. The same format

Figure 4. (A) Standard curves for 2,3,7,8-TCDD using DD1 monoclonal antibody, DD1 enzymatically derived Fab, and recombinant antibody rFab1-1. (B) Standard curves for 2,3,7,8TCDD using DD3 monoclonal antibody, DD3 enzymatically derived Fab, and recombinant antibody rFab3-3.

was employed in the initial development of competition immunoassays using rFab antibodies. Optimum conditions were determined initially by titration of both coating antigen and antibodies in a homologous system to give color development of ∼1.0 absorbance unit. A comparison was made between detection of the polyhistidine tag (i.e., with anti-polyhistidine antibody) and detection of the rFab antibody (i.e., without antipolyhistidine antibody) in the competitive ELISA. A ∼3-fold improvement was achieved by use of direct detection of rFab fragments. Because our coating antigen was >10 years old, all antibodies were purified and assayed concurrently to ensure a direct comparison of the parent, enzymatically derived Fab, and rFab antibodies. Standard curves were performed at least three times to provide sufficient reproducibility, and the ELISA data shown in this paper were calculated using three sets of data obtained on three different days. The standard curves using monoclonal antibody, enzymatically derived Fab, and recombinant Fab are shown in partsA (for DD1) and B (for DD3) of Figure 4. The IC50 using the DD1 monoclonal antibody was 16.3 ( 4.2 ng/ mL, which was comparable to the published data (Stanker et al., 1987). The standard curves for monoclonal antibody, enzymatically derived Fab, and rFab antibodies derived from DD1 were within the standard deviation of each other, indicating they exhibit a similar sensitivity for the competitor. The IC50 values for

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Table 1. Cross-Reactivity of MAb, Enzymatically Derived Fab, and Recombinant Fab Fragments for Dioxin, Biphenyl, and Other Related Compounds cross-reaction (%) DD1 compound dioxin 2,3,7,8-TCDD dibenzodioxin PCB 3,3′,4,4′-TCBP other compounds 2,4,5-trichlorophenol 2,5-dichloronitrobenzene hexachlorocyclohexane (γ-isomer) hexachlorocyclohexane (δ-isomer)

IC50a 0.015 nic

Fab-DD1 CRb

100

nid

rFab1-1

IC50a

CRb

IC50a

0.025 nic

100

0.01 nic

nid

CRb 100

nid

DD3

Fab-DD3

IC50a

CRb

IC50a

CRb

0.007 nic

100

0.01 nic

100

rFab3-3 IC50a 0.01 nic

nid

nid

nid

CRb 100

510 90 nie

0.003 0.02

250 75 nie

0.01 0.03

300 42 nie

0.003 0.02

nie nie nie

nie nie nie

>1000 >1000 nie